Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.
The malaria parasite is considered to be one of the single most serious infectious agents in the world, infecting 5% of the global population and causing serious mortality and morbidity to sensitive populations and hampering socio-economic development.
Severe malaria infection shares several clinical features in common with bacterial septic shock. In both conditions, the excess production by macrophages of pro-inflammatory cytokines such as Tumor Necrosis Factor (TNF), Interleukin-1 (IL-1) and IL-6 occurs in response to malaria “toxins” and lipopolysaccharide (LPS), respectively, leading to complications such as fever and hyperpyrexia, leukopenia, thrombocytopenia, hypotension, disseminated intravascular coagulation, leukocyte infiltration, vascular permeability and multi-organ inflammation, which may lead eventually to death. Thus, many signs, symptoms and syndromes in acute and severe malaria infection result from the activity of a parasite “toxin” released into the circulation during the blood-stage developmental cycle of the infection.
GPI has been identified as a candidate toxin of parasite origin (Schofield, L. and Hackett, F. (1993) Journal of Experimental Medicine 177:145-153 and Tachado, S. D., Gerold, P., Schwarz, R., Novakovic, S., McConville, M., and Schofield, L. (1997) Proceedings of the National Academy of Sciences USA 94:4022-4027). The structure of the molecule has been elucidated (Gerold, P., Dieckman-Schuppert A. and Schwarz, R. T. (1992) Bio. Soc. Trans. 29:297 and Gerold, P., Schofield, L., Brackman, M., Holder, A. A., Schwarz, R. T. (1996) Mol. Biochem. Parasitol 75:131) and it comprises a lipidic domain and a glycan domain. Intact GPI occurs in two closely related forms, Pfglα (NH—CH2—CH2—PO4-(Manα1-2)-6Manα1-2Manα1-6Manα1-4GlcN-H2α1-6(myristoyl)-myo-Inositol-1-PO4-dipalmitoylglycerol), and Pfglβ (NH—CH2—CH2—PO4-6Manα1-2Manα1-6Manα1,4GlcN-H2α 1-6(myristoyl)-myo-Inositol-1-PO4-dipalmitoylglycerol).
The parasite derived GPI molecule regulates host cell function and gene expression in various tissues by activating endogenous GPI-based signal transduction pathways, involving hydrolysis into second messengers and the activation of both tyrosine and serine/threonine kinases. This leads to the activation of the NFκB/c-rel family of transcription factors, which regulate the expression of numerous pro-inflammatory loci implicated in malarial pathology, such as TNF, IL-1, iNOS and ICAM-1.
The toxin theory of malarial pathogenesis can be ascribed to Camillo Golgi, in 1886, who hypothesized that the proximal cause of the febrile paroxysm was a released toxin of parasite origin (Golgi, C. (1886) Arch. Sci. Med. (Torino) 10:109). Clark proposed that the systemic inflammation of malaria infection resulted from a functional malarial endotoxin, and suggested that this agent exerts systemic effects largely through the induction of endogenous pyrogens of host origin. Clark correctly identified TNF as a major host mediator of disease (Clark, I. A. (1978) Lancet 2:75 and Clark, I. A., Virelizier, J.-L., Carswell, E. A., and Wood, P. R. (1981) Infect. Immun. 32:1058). Consequently, the production of this and related pyrogenic cytokines (IL-1, IL-6) from monocyte/macrophages is often taken as a useful surrogate marker for the initiation of pathological processes in malaria infection. John Playfair and his colleagues extended this work to show that crude extracts of rodent malaria parasites could induce macrophages to secrete TNF in vitro (Bate, C. A., Taverne, J., and Playfair, J. H. (1988) Immunology 64:227 and Bate, C. A., Taverne, J., and Playfair, J. H. (1989) Immunology 66:600) and inferred that the toxin included a phospholipid moiety. Nonetheless, prior to the advent of the present invention, the specific biochemical identity of the parasite toxin, and its mechanism of action, have remained obscure.
In work leading up to the present invention, the inventors investigated the use of portions of GPI to induce protective immunity against malarial pathology. The inventors have surprisingly discovered that GPI portions which exclude the lipidic domain induce protective immunity whereas portions carrying the lipidic domain do not.
In still another surprising aspect, it has been determined that the above-described GPI portions can be effectively utilised to facilitate the qualitative and/or quantitative analysis of the immune response which has been generated to a parasite, in particular Plasmodium, to parasite pathology, such as malarial pathology. Still further, the analysis of the immune response in this unique and surprisingly effective antigen-based manner provides a means of facilitating epitope specific screening or the rational design of functionally effective immunogenic molecules. This aspect of the present invention has been particularly facilitated by the successful generation of synthetic GPI molecules.